Elsevier

Cell Calcium

Volume 36, Issue 6, December 2004, Pages 459-467
Cell Calcium

The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells

https://doi.org/10.1016/j.ceca.2004.04.004Get rights and content

Abstract

Calcium entry is a component of the processes regulating the proliferative phenotype of some types of cancer. In non-excitable cells, capacitative calcium entry (CCE) and non-capacitative calcium entry (NCCE) are thought to be the main pathways of Ca2+ influx into cells. Thus, blocking calcium entry may prevent normal and pathological cell proliferation and there is evidence to suggest that molecules blocking calcium entry also have antiproliferative properties. Carboxyamidotriazole (CAI), a novel inhibitor of the non-voltage-dependent calcium entry has been shown to have such properties in model systems in vitro and in vivo. We used Hep G2 and Huh-7 human hepatoma cells to investigate the effects of calcium entry blockers on cell proliferation. CAI (10 μM) and 2-APB (20 μM) completely blocked CCE in thapsigargin-treated Huh-7, and CAI and 2-APB inhibited cell proliferation with IC50 of 4.5 and 43 μM, respectively. The plateau phase of the [Ca2+]i increases triggered by 10% FCS were abolished in the absence of external Ca2+ and in the presence of CAI or 2-APB. We, therefore, suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.

Introduction

Calcium plays a key role in cell physiology. The stimulation of liver cells by growth factors results in rapid and sustained increases in intracellular free calcium concentrations, [Ca2+]i [1], [2] leading to the activation of several transcription factors including c-jun, c-myc, and c-fos and of cell cycle-associated cyclin expression [3], [4]. In turn, these transcription factors regulate the expression of several inducible genes mediating diverse programs including cell proliferation, cell differentiation and cell death [5], [6].

Intracellular calcium signals in non-excitable cells, such as liver cells, involve the rapid, transient InsP3-mediated release of calcium from stores, leading to a decrease in the calcium concentration of the ER lumen, followed by the activation of plasma membrane calcium channels to generate a sustained influx of calcium [7]. This type of calcium influx is called capacitative or store-operated calcium entry (CCE) [8]. It is thought to be an essential component of the long-term responses of the cell, including proliferation [9], [10]. Submaximal agonist concentrations unable to trigger intracellular Ca2+ store depletion have been associated with another Ca2+ entry pathway—non-capacitative calcium entry (NCCE)—activated by diacylglycerol [11] or arachidonic acid [12]. It has been known for more than 30 years that the proliferation and growth of normal vertebrate cells are dependent on the physiological concentration of extracellular calcium [13], [14]. Calcium entry is required at various stages of the cell cycle [15], [16] but little is currently known about the nature of the calcium channels involved in this process in non-excitable cells such as liver cells. There is growing evidence to suggest that CCE amplitude is related to cell proliferation in several cell types [10], [17], [18], [19]. The blocking of calcium influx may prevent normal and pathological cell proliferation and there is some evidence to suggest that molecules blocking calcium entry also have antiproliferative properties [20], [21]. Carboxyamidotriazole (CAI), a novel inhibitor of non-voltage-dependent calcium entry, has been shown to have such properties in model systems in vitro and in vivo [16]. In this study, we used two human hepatoma cell lines, Hep G2 and Huh-7, to investigate the relationship between cell proliferation and CCE, using CAI and a known CCE blocker, 2-APB. We suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.

Section snippets

Cell culture

Hep G2 and Huh-7 human hepatoma cells were provided by Dr. Doris Cassio (INSERM U442, Orsay, France). Cells were cultured in complete medium (Dulbecco’s MEM with Glutamax-I supplemented with fetal calf serum (FCS, 10%), penicillin (200,000 U/ml), and streptomycin (100 μg/ml)), at 37 °C, in an atmosphere containing 5% CO2. Hep G2 and Huh-7 cells were plated at densities of 50,000 and 10,000 cells/cm2, respectively, and cultured to about 80% confluence.

Cell proliferation

Hep G2 and Huh-7 cells were plated in 24-well

Calcium measurements

Cell suspensions: Huh-7 cells (5 × 106 cells/ml) were loaded at 37 °C for 45 min with 2 μM Fura-2/AM in complete DMEM culture medium. Cells were treated with trypsin and washed once by centrifugation at 50 × g for 1 min in the same medium. Cell pellets were resuspended in a medium containing 116 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1 mM NaH2PO4, 5 mM NaHCO3, 0.1 mM EGTA, 20 mM HEPES, pH 7.3, with or without 1.8 mM CaCl2, as indicated. Cell pellets were transferred to a quartz cuvette and placed in the light

Effects of CAI and 2-APB on human hepatoma cell line proliferation

The proliferation of human hepatoma cells was assessed by measuring the incorporation of [3H]thymidine. Cells were first incubated for 24 h in the presence of 10% FCS; the external medium was replaced by fresh medium supplemented with various concentrations of CAI or 2-APB and the cells were incubated for a further 24 h (Fig. 1). CAI blocks the incorporation of [3H]thymidine in Hep G2 and Huh-7 cells, with IC50 of 0.7 and 4.5 μM, respectively (Fig. 1A and B). The incorporation of [3H]thymidine was

Discussion

The overexpression of transient receptor potential (TRP) channels results in increases in CCE and cell proliferation [10], [18], [19], suggesting that these two cellular processes are intimately related. Our results showed that blocking CCE with CAI and 2-APB inhibited proliferation in the Hep G2 and Huh-7 cell lines. 2-APB and CAI inhibit CCE and cell proliferation with similar potency in human hepatoma cell lines, suggesting that sustained increase in [Ca2+]i is required to activate the genes

Acknowledgements

We thank Josiane Simon for technical assistance and Julie Sappa (Alex Edelmann & Associates, Paris, France) for editing the manuscript. This work was supported by grants from Association pour la Recherche contre le Cancer (ARC no. 5241, 5621 and 4615) and Comité de l’Essonne de la Ligue contre le Cancer.

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    Present address: INSERM U526, Faculté de Médecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex 02, France.

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